1. Field of the Invention
The present invention relates to disulfide-stabilized (ds) recombinant polypeptide molecules, such as the variable region of an antibody molecule, which have the binding ability and specificity for another peptide. Methods of producing these molecules and nucleic acid sequences encoding these molecules are also described.
2. In the Background
Antibodies are molecules that recognize and bind to a specific cognate antigen. Numerous applications of hybridoma-produced monoclonal antibodies for use in clinical diagnosis, treatment, and basic scientific research have been described. Clinical treatments of cancer, viral and microbial infections, B cell immunodeficiencies, and other diseases and disorders of the immune system using monoclonal antibodies appear promising. Fv fragments of immunoglobulins are considered the smallest functional component of antibodies required for high affinity binding of antigen. Their small size makes them potentially more useful than whole antibodies for clinical applications like imaging tumors and directing recombinant immunotoxins to tumors since size strongly influences tumor and tissue penetration.
Fv fragments are heterodimers of the variable heavy chain domain (VH) and the variable light chain domain (VL). The heterodimers of heavy and light chain domains that occur in whole IgG, for example, are connected by a disulfide bond. The Fv fragments are not and therefore Fvs alone are unstable. Glockshuber et al., Biochemistry 29:1362-1367 (1990). Recombinant Fvs which have VH and VL connected by a peptide linker are typically stable, see, for example, Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988) and Bird et al., Science 242:423-426 (1988). These are single chain Fvs which have been found to retain specificity and affinity and have been shown to be useful for imaging tumors and to make recombinant immunotoxins, for tumor therapy for example. However, researchers have found that some of the single chain Fvs have a reduced affinity for antigen and the peptide linker can interfere with binding.
Another approach to stabilize the Fvs was attempted by glockshuber et al., supra. Disulfide bonds were placed in the complementarity determining regions (CDR) of an antibody whose structure was known in a manner that had limited or no effect on ligand binding. This approach is problematic for stabilizing other Fvs with unknown structures because the structure of each CDR region changes from one antibody to the next and because disulfide bonds that bridge CDRs will likely interfere with antigen binding. Thus, it would be desirable to have alternative means to stabilize the Fv portions of an antibody of interest which would allow the affinity for the target antigen to be maintained.
The invention relates to a polypeptide specifically binding a ligand, wherein the polypeptide comprises a first variable region of a ligand binding moiety bound through a disulfide bond to a second separate variable region of the ligand binding moiety, the bond connecting framework regions of the first and second variable regions. The polypeptide may be conjugated to a radioisotope, an enzyme, a toxin, or a drug or may be recombinantly fused to a toxin, enzyme or a drug, for example. Nucleic acid sequences coding the polypeptides and pharmaceutical compositions containing them are also disclosed.
The polypeptide is preferably one, wherein the first variable region is a light chain variable region of an antibody and the second variable region is a heavy chain variable region of the antibody. The polypeptide may also be one, wherein the first variable region is an xcex1 variable chain region of a T cell receptor and the second variable region is a xcex2 variable chain region of the T cell receptor.
Methods for producing a disulfide stabilized polypeptide of a ligand binding moiety having a two variable regions are also disclosed comprising the following steps:
(a) mutating a nucleic acid for the first variable region so that cysteine is encoded at position 42, 43, 44, 45 or 46, and mutating a nucleic acid sequence for the second variable region so that cysteine is encoded at position 103, 104, 105, or 106, such positions being determined in accordance with the numbering scheme published by Kabat and Wu, corresponding to a light chain and a heavy chain region, respectively, of an antibody; or
(b) mutating a nucleic acid for the first variable region so that cysteine is encoded at position 43, 44, 45, 46 or 47 and mutating a nucleic acid for the second variable region so that cysteine is encoded at position 98, 99, 100, or 101 such positions being determined in accordance with the numbering scheme published by Kabat and Wu, corresponding to a heavy chain or a light chain region respectively of an antibody; then
(c) expressing the nucleic acid for the first variable region and the nucleic acid for the second variable region in an expression system; and
(d) recovering the polypeptide having a binding affinity for the antigen.
The invention provides an alternative means to recombinant Fvs which have VH and VL connected by a peptide linker. Though such recombinant single chain Fvs are typically stable and specific, some have a reduced affinity for antigen and the peptide linker can interfere with binding. A means to produce recombinant Fv polypeptides that are stabilized by a disulfide bond located in the conserved regions of the Fvb fragment and compositions that include these, such as immunotoxins, are also described.
The clinical administration of the small polypeptides of the invention affords a number of advantages over the use of larger fragments or entire antibody molecules. The polypeptides of this invention in preferred forms have greater stability due to the additional disulfide bond. Due to their small size they also offer fewer cleavage sites to circulating proteolytic enzymes resulting in greater stability. They reach their target tissue more rapidly, and are cleared more quickly from the body. They also have reduced immunogenicity. In addition, their small size facilitates specific coupling to other molecules in drug targeting and imaging applications.
The invention also provides a means of stabilizing the antigen-binding portion (the V domain) of the T cell receptors, by connecting the xcex1 and xcex2 chains of the V domain by an inter-chain disulfide bond. Such stabilization of the V domain will help isolate and purify this fragment in soluble form. The molecule can then be used in applications similar to those of other Fvs. They can be used in diagnostic assays for tumor cells or for detection of immune-based diseases such as autoimmune diseases and AIDS. They may also have therapeutic use as a target for tumor cells or as a means to block undesirable immune responses in autoimmune diseases, or other immune-based disease.